蟾毒灵联合吉非替尼对肺癌细胞H1975的影响及机制研究

目的观察蟾毒灵联合吉非替尼对肺癌细胞H1975生长的影响，探讨蟾毒灵增强吉非替尼抗肿瘤作用的可能机制。方法应用MTT法检测蟾毒灵（1～100 nmol/L）、吉非替尼（0.1～20 μmol/L）及两药联合干预对H1975细胞增殖的抑制作用；流式细胞术观察蟾毒灵（10 nmol/L）、吉非替尼（1 μmol/L）及联合用药对H1975细胞凋亡的影响；Western blot检测单独用药或联合用药对表皮生长因子受体（epidermal growth factor receptor，EGFR）、Met信号通路相关蛋白表达的影响。结果 MTT结果显示，吉非替尼浓度超过5 μmol/L时才见到其对H1975细胞生长的抑制作用；蟾毒灵与吉非替尼联合后，可明显抑制肿瘤细胞的生长。流式细胞术结果显示，蟾毒灵联合吉非替尼干预后肿瘤细胞凋亡率为（61.64±5.61）%，明显高于单用蟾毒灵的（18.34±3.42）%和单用吉非替尼的（7.32±1.08）%，差异有统计学意义（P<0.01）。Western blot结果显示，蟾毒灵联合吉非替尼能明显下调H1975细胞p-EGFR、p-Met、磷酸化蛋白激酶B（p-Akt）、磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）蛋白表达。结论蟾毒灵联合吉非替尼可显著抑制H1975细胞的增殖，促使肿瘤细胞凋亡；其抗肿瘤活性的机制可能与阻断EGFR－磷酯酰肌醇－3激酶（PI3k）/Akt信号通路有关。

英文摘要:

Objective To observe the effect of bufalin combined Gefitinib on lung cancer H1975 cells， and to explore its potential mechanisms for anti-tumor. Methods The cytostatic effects of bufalin （1－100 nmol/L）， gefitinib （0.1－20 μmol/L）， and bufalin plus gefitinib on H1975 cells were evaluated by MTT assay. Their effects on apoptosis of H1975 cells were determined by flow cytometry （FCM）. Their effects on expressions of epidermal growth factor receptor （EGFR） and Met signal pathway related proteins in H1975 cells were detected by Western blot. Results Results of MTT assay showed that gefitinib over 5 μmol/L could inhibit H1975 cells. But combined therapy of bufalin and gefitinib could potently inhibit the growth of H1975 cells. Results of FCM showed the apoptotic rate was 61.64%±5.61% in the bufalin plus gefitinib group， obviously higher than that of the bufalin group （18.34%±3.42%） and the gefitinib group （7.32%±1.08%）， showing statistical difference （P<0.01）. Results of Western blot showed the protein expressions of p-EGFR， p-Met， p-Akt， and p-mTOR in H1975 cells could be markedly down-regulated by bufalin plus gefitinib. Conclusions Combination of bufalin and gefitinib potently inhibited the growth of H1975 cells， and induced cell apoptosis. The potential mechanism for anti-tumor might be involved in blocking EGFR-PI3k/Akt pathway.

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